Glutathione Protects other Cellular Thiols against Oxidation by CuII-Dp44mT.

Cu-thiosemicarbazones have been intensively investigated for their application in cancer therapy or as antimicrobials. Copper(II)-di-2-pyridylketone-4,4-dimethyl-thiosemicarbazone (CuII-Dp44mT) showed anticancer activity in the submicromolar concentration range in cell culture. The interaction of CuII-Dp44mT with thiols leading to their depletion or inhibition was proposed to be involved in this activity. Indeed, CuII-Dp44mT can catalyze the oxidation of thiols although with slow kinetics. The present work aims to obtain insights into the catalytic activity and selectivity of CuII-Dp44mT toward the oxidation of different biologically relevant thiols. Reduced glutathione (GSH), L-cysteine (Cys), N-acetylcysteine (NAC), D-penicillamine (D-Pen), and the two model proteins glutaredoxin (Grx) and thioredoxin (Trx) were investigated. CuII-Dp44mT catalyzed the oxidation of these thiols with different kinetics, with rates in the following order D-Pen>Cys≫NAC>GSH and Trx>Grx. CuII-Dp44mT was more efficient than CuII chloride for the oxidation of NAC and GSH, but not D-Pen and Cys. In mixtures of biologically relevant concentrations of GSH and either Cys, Trx, or Grx, the oxidation kinetics and spectral properties were similar to that of GSH alone, indicating that the interaction of these thiols with CuII-Dp44mT is dominated by GSH. Hence GSH could protect other thiols against potential deleterious oxidation by CuII-Dp44mT.

Association of Urinary N7-(1-hydroxyl-3-buten-1-yl) Guanine (EB-GII) Adducts and Butadiene-Mercapturic Acids with Lung Cancer Development in Cigarette Smokers.

Approximately 10% of smokers will develop lung cancer. Sensitive predictive biomarkers are needed to identify susceptible individuals. 1,3-Butadiene (BD) is among the most abundant tobacco smoke carcinogens. BD is metabolically activated to 3,4-epoxy-1-butene (EB), which is detoxified via the glutathione conjugation/mercapturic acid pathway to form monohydroxybutenyl mercapturic acid (MHBMA) and dihydroxybutyl mercapturic acid (DHBMA). Alternatively, EB can react with guanine nucleobases of DNA to form N7-(1-hydroxyl-3-buten-1-yl) guanine (EB-GII) adducts. We employed isotope dilution LC/ESI-HRMS/MS methodologies to quantify MHBMA, DHBMA, and EB-GII in urine of smokers who developed lung cancer (N = 260) and matched smoking controls (N = 259) from the Southern Community Cohort (white and African American). The concentrations of all three biomarkers were significantly higher in smokers that subsequently developed lung cancer as compared to matched smoker controls after adjusting for age, sex, and race/ethnicity (p < 0.0001 for EB-GII, p < 0.0001 for MHBMA, and p = 0.0007 for DHBMA). The odds ratio (OR) for lung cancer development was 1.63 for MHBMA, 1.37 for DHBMA, and 1.97 for EB-GII, with a higher OR in African American subjects than in whites. The association of urinary EB-GII, MHBMA, and DHBMA with lung cancer status did not remain upon adjustment for total nicotine equivalents. These findings reveal that urinary MHBMA, DHBMA, and EB-GII are directly correlated with the BD dose delivered via smoking and are associated with lung cancer risk.

TRIM6 Promotes ROS-Mediated Inflammasome Activation and Pyroptosis in Renal Tubular Epithelial Cells via Ubiquitination and Degradation of GPX3 Protein.

BACKGROUND: Pyroptosis is a critical form of cell death during the development of chronic kidney disease (CKD). Tripartite motif 6 (TRIM6) is an E3-ubiquitin ligase that participates in the progression renal fibrosis (RF). The aim of this study was to investigate the roles of TRIM6 and Glutathione peroxidase 3 (GPX3) in oxidative stress-induced inflammasome activation and pyroptosis in Ang-II treated renal tubular epithelial cells. METHODS: To study its role in RF, TRIM6 expression was either reduced or increased in human kidney-2 (HK2) cells using lentivirus, and Ang-II, NAC and BMS-986299 were served as reactive oxygen species (ROS) inducer, ROS scavenger and NLRP3 agonist respectively. Pyroptosis and mitochondrial ROS were measured by flow cytometry. The levels of malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) were determined using commercial kits, while the levels of IL-1ß, IL-18, IL-6, and tumor necrosis factor-α (TNF-α) were determined by Enzyme-Linked Immunosorbent Assay (ELISA). Co-immunoprecipitation (Co-IP) assay was used to evaluate the interaction between TRIM6 and GPX3. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot were used to measure mRNA and protein expression, respectively. RESULTS: Treatment with Angiotensin II (Ang II) increased the protein and mRNA levels of TRIM6 in HK2 cells. Ang II also increased mitochondrial ROS production and the malondialdehyde (MDA) level, but decreased the levels of GSH and SOD. In addition, Ang II enhanced HK2 cell pyroptosis, increased the levels of IL-1ß, IL-18, IL-6, and TNF-α, and promoted the expression of active IL-1ß, NLRP3, caspase-1, and GSDMD-N proteins. These effects were reversed by knockdown of TRIM6 and by treatment with N-acetyl-L-cysteine (NAC), a ROS scavenger. BMS-986299, an NLRP3 agonist treatment, did not affect ROS production in HK2 cells exposed to Ang II combined with NAC, but cell pyroptosis and inflammation were aggravated. Moreover, the overexpression of TRIM6 in HK2 cells resulted in similar effects to Ang II. NAC and GPX3 overexpression in HK2 cells could reverse ROS production, inflammation, and pyroptosis induced by TRIM6 overexpression. TRIM6 overexpression decreased the GPX3 protein level by promoting its ubiquitination, without affecting the GPX3 mRNA level. Thus, TRIM6 facilitates GPX3 ubiquitination, contributing to increased ROS levels and pyroptosis in HK2 cells. CONCLUSIONS: TRIM6 increases oxidative stress and promotes the pyroptosis of HK2 cells by regulating GPX3 ubiquitination. These findings could contribute to the development of novel drugs for the treatment of RF.

Acetylated oligopeptide and N-acetylcysteine protect against iron overload-induced dentate gyrus hippocampal degeneration through upregulation of Nestin and Nrf2/HO-1 and downregulation of MMP-9/TIMP-1 and GFAP.

Iron accumulation in the brain causes oxidative stress, blood-brain barrier (BBB) breakdown, and neurodegeneration. We examined the preventive effects of acetylated oligopeptides (AOP) from whey protein on iron-induced hippocampal damage compared to N-acetyl cysteine (NAC). This 5-week study used 40 male albino rats. At the start, all rats received 150 mg/kg/day of oral NAC for a week. The 40 animals were then randomly divided into four groups: Group I (control) received a normal diet; Group II (iron overload) received 60 mg/kg/day intraperitoneal iron dextran 5 days a week for 4 weeks; Group III (NAC group) received 150 mg/kg/day NAC and iron dextran; and Group IV (AOP group) received 150 mg/kg/day AOP and iron dextran. Enzyme-linked immunosorbent assay, spectrophotometry, and qRT-PCR were used to measure MMP-9, tissue inhibitor metalloproteinase-1 (TIMP-1), MDA, reduced glutathione (GSH) levels, and nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) gene expression. Histopathological and immunohistochemical detection of nestin, claudin, caspase, and GFAP was also done. MMP-9, TIMP-1, MDA, caspase, and GFAP rose in the iron overload group, while GSH, Nrf2, HO-1, nestin, and claudin decreased. The NAC and AOP administrations improved iron overload-induced biochemical and histological alterations. We found that AOP and NAC can protect the brain hippocampus from iron overload, improve BBB disruption, and provide neuroprotection with mostly no significant difference from healthy controls.

Silver nanoparticles induce endothelial cytotoxicity through ROS-mediated mitochondria-lysosome damage and autophagy perturbation: The protective role of N-acetylcysteine.

Silver nanoparticles (AgNPs) are used increasingly often in the biomedical field, but their potential deleterious effects on the cardiovascular system remain to be elucidated. The primary aim of this study was to evaluate the toxic effects, and the underlying mechanisms of these effects, of AgNPs on human umbilical vein endothelial cells (HUVECs), as well as the protective role of N-acetylcysteine (NAC) against cytotoxicity induced by AgNPs. In this study, we found that exposure to AgNPs affects the morphology and function of endothelial cells which manifests as decreased cell proliferation, migration, and angiogenesis ability. Mechanistically, AgNPs can induce excessive cellular production of reactive oxygen species (ROS), leading to damage to cellular sub-organs such as mitochondria and lysosomes. More importantly, our data suggest that AgNPs causes autophagy defect, inhibits mitophagy, and finally activates the mitochondria-mediated apoptosis signaling pathway and evokes cell death. Interestingly, treatment with ROS scavenger-NAC can effectively suppress AgNP-induced endothelial damage.Our results indicate that ROS-mediated mitochondria-lysosome injury and autophagy dysfunction are potential factors of endothelial toxicity induced by AgNPs. This study may provide new evidence for the cardiovascular toxicity of AgNPs and serve as a reference for the safe use of nanoparticles(NPs) in the future.

Protective effects of l-cysteine and N-acetyl-l-cysteine on boar sperm quality during hypothermic liquid storage with bovine serum albumin as a protectant.

Hypothermic liquid storage at 4-5 °C has emerged as a novel approach for preserving boar semen, offering innovative possibilities for semen preservation. However, this method also presents challenges, including cold shock and excessive reactive oxygen species (ROS) production. Therefore, reducing oxidative damage induced by low temperatures becomes essential while supplementing appropriate protectants. In this study, we investigated the efficacy of Bovine Serum Albumin (BSA) compared to Polyvinylpyrrolidone (PVP) and Skim Milk Powder (SMP) in maintaining boar sperm motility and progressive motility using computer-assisted sperm analysis (CASA). Among the tested concentrations, 4 g/L of BSA exhibited the best protective effect. Subsequently, we supplemented different concentrations of l-cysteine (LC) and N-acetyl-l-cysteine (NAC) as additives in the presence of BSA as a protectant. Our results demonstrated that 1 mmol/L of LC and 0.5 mmol/L of NAC exhibited superior protection of sperm quality compared to other concentrations. Furthermore, the 1 mmol/L LC and 0.5 mmol/L NAC groups showed significantly improved plasma membrane integrity and acrosome integrity compared to the control group. These groups also exhibited enhanced antioxidant capacity, evidenced by increased mitochondrial membrane potential (MMP), ATP production, total superoxide dismutase (T-SOD) activity, total antioxidant capacity (T-AOC), glutathione (GSH), glutathione peroxidase (GSH-PX), and GPX-4 levels. Additionally, they demonstrated decreased reactive oxygen species (ROS) and malondialdehyde (MDA) levels, as well as reduced oxidized glutathione (GSSG) and glutathione reductase (GR) levels. Furthermore, LC and NAC treatment enhanced AMP-activated protein kinase (AMPK) phosphorylation. However, inhibiting AMPK using compound C did not inhibit the protective effects of LC and NAC on low-temperature preserved boar sperm. These findings suggest that 4 g/L BSA can serve as an effective protectant for hypothermic liquid storage of boar semen. Additionally, LC and NAC supplementation reduces oxidative damage by enhancing antioxidant capacity rather than through AMPK-mediated ATP supplementation. These results contribute to advancing the application of LC and NAC in hypothermic liquid storage of boar semen.

The antioxidant N-acetyl cysteine inhibits cytokine and prostaglandin release in human fetal membranes stimulated ex vivo with lipoteichoic acid or live group B streptococcus.

BACKGROUNDS: Infection during pregnancy is a significant public health concern due to the increased risk of adverse birth outcomes. Group B Streptococcus or Streptococcus agalactiae (GBS) stands out as a major bacterial cause of neonatal morbidity and mortality. We aimed to explore the involvement of reactive oxygen species (ROS) and oxidative stress pathways in pro-inflammatory responses within human fetal membrane tissue, the target tissue of acute bacterial chorioamnionitis. METHODS: We reanalyzed transcriptomic data from fetal membrane explants inoculated with GBS to assess the impact of GBS on oxidative stress and ROS genes/pathways. We conducted pathway enrichment analysis of transcriptomic data using the Database for Annotation, Visualization and Integrated Discovery (DAVID), a web-based functional annotation/pathway enrichment tool. Subsequently, we conducted ex vivo experiments to test the hypothesis that antioxidant treatment could inhibit pathogen-stimulated inflammatory responses in fetal membranes. RESULTS: Using DAVID analysis, we found significant enrichment of pathways related to oxidative stress or ROS in GBS-inoculated human fetal membranes, for example, "Response to Oxidative Stress" (FDR = 0.02) and "Positive Regulation of Reactive Oxygen Species Metabolic Process" (FDR = 2.6*10-4 ). There were 31 significantly changed genes associated with these pathways, most of which were upregulated after GBS inoculation. In ex vivo experiments with choriodecidual membrane explants, our study showed that co-treatment with N-acetylcysteine (NAC) effectively suppressed the release of pro-inflammatory cytokines (IL-6, IL-8, TNF-α) and prostaglandin PGE2, compared to GBS-treated explants (p < .05 compared to GBS-treated samples without NAC co-treatment). Furthermore, NAC treatment inhibited the release of cytokines and PGE2 stimulated by lipoteichoic acid (LTA) and lipopolysaccharide (LPS) in whole membrane explants (p < .05 compared to LTA or LPS-treated samples without NAC co-treatment). CONCLUSIONS: Our study sheds light on the potential roles of ROS in governing the innate immune response to GBS infection, offering insights for developing strategies to mitigate GBS-related adverse outcomes.

Akt Signaling and Nitric Oxide Synthase as Possible Mediators of the Protective Effect of N-acetyl-L-cysteine in Prediabetes Induced by Sucrose.

The aim of this work was to evaluate possible mechanisms involved in the protective effect of N-acetyl-L-cysteine (NAC) on hepatic endocrine-metabolic, oxidative stress, and inflammatory changes in prediabetic rats. For that, normal male Wistar rats (60 days old) were fed for 21 days with 10% sucrose in their drinking water and 5 days of NAC administration (50 mg/kg, i.p.) and thereafter, we determined: serum glucose, insulin, transaminases, uric acid, and triglyceride levels; hepatic fructokinase and glucokinase activities, glycogen content, lipogenic gene expression; enzymatic and non-enzymatic oxidative stress, insulin signaling pathway, and inflammatory markers. Results showed that alterations evinced in sucrose-fed rats (hypertriglyceridemia, hyperinsulinemia, and high liver fructokinase activity together with increased liver lipogenic gene expression and oxidative stress and inflammatory markers) were prevented by NAC administration. P-endothelial nitric oxide synthase (P-eNOS)/eNOS and pAKT/AKT ratios, decreased by sucrose ingestion, were restored after NAC treatment. In conclusion, the results suggest that NAC administration improves glucose homeostasis, oxidative stress, and inflammation in prediabetic rats probably mediated by modulation of the AKT/NOS pathway. Administration of NAC may be an effective complementary strategy to alleviate or prevent oxidative stress and inflammatory responses observed in type 2 diabetes at early stages of its development (prediabetes).

N-acetylcysteine alleviated tris(2-chloroisopropyl) phosphate-induced sperm motility decline and functional dysfunction in mice through reversing oxidative stress and DNA damage.

The decline in male fertility caused by environmental pollutants has attracted worldwide attention nowadays. Tris(2-chloroisopropyl) phosphate (TCPP) is a chlorine-containing organophosphorus flame retardant applied in many consumer products and has multiple side effects on health. However, whether TCPP impairs spermatogenesis remains unclear. In this study, we found that TCPP reduced the sperm motility and blastocyst formation, inhibited proliferation and induced apoptosis in mice testes and spermatocyte cell line GC-2. Moreover, TCPP induced imbalance of oxidant and anti-oxidant, DNA damage and mitochondrial dysfunction, thus induced abnormal spermatogenesis. In this process, p53 signaling pathway was activated and N-acetylcysteine treatment partially alleviated the side effects of TCPP, including decrease of sperm motility, activation of p53 signaling pathway and DNA damage. Finally, our study verified that TCPP elevated reactive oxygen species (ROS), decreased mitochondrial membrane potential and induced apoptosis in human semen samples. Overall, ROS mediated TCPP-induced germ cell proliferation inhibition and apoptosis, which finally led to the decline of sperm motility.

N-acetylcysteine and Hydroxychloroquine Ameliorate ADMA-Induced Fetal Growth Restriction in Mice via Regulating Oxidative Stress and Autophagy.

Fetal growth restriction (FGR) seriously threatens perinatal health. The main cause of FGR is placental malperfusion, but the specific mechanism is still unclear, and there is no effective treatment for FGR. We constructed a FGR mouse model by adding exogenous asymmetric dimethylarginine (ADMA) through in vivo experiments and found that ADMA could cause placental dysplasia and induce the occurrence of FGR. Compared with the control group, reactive oxygen species (ROS) production in the placenta was increased in mice with FGR, and the expression of autophagy-related proteins p-AKT/AKT, p-mTOR/mTOR, and P62 was significantly decreased, while the expression of Beclin-1 and LC3-II was significantly increased in the FGR group. Furthermore, ADMA had a favorable effect in promoting the formation of autophagosomes. Hydroxychloroquine (HCQ) and N-acetylcysteine (NAC) improved ADMA-induced disorders of placental development and alleviated ADMA-induced FGR. This study found that ADMA could cause excessive autophagy of trophoblasts by increasing the level of oxidative stress, ultimately leading to the occurrence of FGR, and HCQ and NAC had therapeutic effects on ADMA-induced FGR.

N-Acetylcysteine Alleviates Phenylephrine-Induced Cardiomyocyte Dysfunction via Engaging PI3K/AKT Signaling Pathway.

BACKGROUND: Increased reactive oxygen species (ROS) and oxidative stress response lead to cardiomyocyte hypertrophy and apoptosis, which play crucial roles in the pathogenesis of heart failure. The purpose of current research was to explore the role of antioxidant N-acetylcysteine (NAC) on cardiomyocyte dysfunction and the underlying molecular mechanisms. METHODS AND RESULTS: Compared with control group without NAC treatment, NAC dramatically inhibited the cell size of primary cultured neonatal rat cardiomyocytes (NRCMs) tested by immunofluorescence staining and reduced the expression of representative markers associated with hypertrophic, fibrosis and apoptosis subjected to phenylephrine administration examined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. Moreover, enhanced ROS expression was attenuated, whereas activities of makers related to oxidative stress response examined by individual assay Kits, including total antioxidation capacity (T-AOC), glutathione peroxidase (GSH-Px), and primary antioxidant enzyme Superoxide dismutase (SOD) were induced by NAC treatment in NRCMs previously treated with phenylephrine. Mechanistically, we noticed that the protein expression levels of phosphorylated phosphatidylinositol 3-kinase (PI3K) and AKT were increased by NAC stimulation. More importantly, we identified that the negative regulation of NAC in cardiomyocyte dysfunction was contributed by PI3K/AKT signaling pathway through further utilization of PI3K/AKT inhibitor (LY294002) or agonist (SC79). CONCLUSIONS: Collected, NAC could attenuate cardiomyocyte dysfunction subjected to phenylephrine, partially by regulating the ROS-induced PI3K/AKT-dependent signaling pathway.

Lack of mitochondrial Cyp2E1 drives acetaminophen-induced ER stress-mediated apoptosis in mouse and human kidneys: Inhibition by 4-methylpyrazole but not N-acetylcysteine.

Acetaminophen (APAP) overdose causes liver injury and acute liver failure, as well as acute kidney injury, which is not prevented by the clinical antidote N-acetyl-L-cysteine (NAC). The absence of therapeutics targeting APAP-induced nephrotoxicity is due to gaps in understanding the mechanisms of renal injury. APAP metabolism through Cyp2E1 drives cell death in both the liver and kidney. We demonstrate that Cyp2E1 is localized to the proximal tubular cells in mouse and human kidneys. Virtually all the Cyp2E1 in kidney cells is in the endoplasmic reticulum (ER), not in mitochondria. By contrast, hepatic Cyp2E1 is in both the ER and mitochondria of hepatocytes. Consistent with this subcellular localization, a dose of 600 mg/kg APAP in fasted C57BL/6J mice induced the formation of APAP protein adducts predominantly in mitochondria of hepatocytes, but the ER of the proximal tubular cells of the kidney. We found that reactive metabolite formation triggered ER stress-mediated activation of caspase-12 and apoptotic cell death in the kidney. While co-treatment with 4-methylpyrazole (4MP; fomepizole) or the caspase inhibitor Ac-DEVD-CHO prevented APAP-induced cleavage of procaspase-12 and apoptosis in the kidney, treatment with NAC had no effect. These mechanisms are clinically relevant because 4MP but not NAC also significantly attenuated APAP-induced apoptotic cell death in primary human kidney cells. We conclude that reactive metabolite formation by Cyp2E1 in the ER results in sustained ER stress that causes activation of procaspase-12, triggering apoptosis of proximal tubular cells, and that 4MP but not NAC may be an effective antidote against APAP-induced kidney injury.

Glycine and N-Acetylcysteine (GlyNAC) Combined with Body Weight Support Treadmill Training Improved Spinal Cord and Skeletal Muscle Structure and Function in Rats with Spinal Cord Injury.

Skeletal muscle atrophy is a frequent complication after spinal cord injury (SCI) and can influence the recovery of motor function and metabolism in affected patients. Delaying skeletal muscle atrophy can promote functional recovery in SCI rats. In the present study, we investigated whether a combination of body weight support treadmill training (BWSTT) and glycine and N-acetylcysteine (GlyNAC) could exert neuroprotective effects, promote motor function recovery, and delay skeletal muscle atrophy in rats with SCI, and we assessed the therapeutic effects of the double intervention from both a structural and functional viewpoint. We found that, after SCI, rats given GlyNAC alone showed an improvement in Basso-Beattie-Bresnahan (BBB) scores, gait symmetry, and results in the open field test, indicative of improved motor function, while GlyNAC combined with BWSTT was more effective than either treatment alone at ameliorating voluntary motor function in injured rats. Meanwhile, the results of the skeletal muscle myofiber cross-sectional area (CSA), hindlimb grip strength, and acetylcholinesterase (AChE) immunostaining analysis demonstrated that GlyNAC improved the structure and function of the skeletal muscle in rats with SCI and delayed the atrophication of skeletal muscle.

Essential Amino Acid Starvation-Induced Oxidative Stress Causes DNA Damage and Apoptosis in Murine Osteoblast-like Cells.

Intracellular nutrient metabolism, particularly the metabolism of essential amino acids (EAAs), is crucial for cellular functions, including energy production and redox homeostasis. An EAA deficiency can lead to cellular dysfunction and oxidative stress. This study explores the mechanisms underlying cellular responses to EAA starvation, focusing on ROS-induced DNA damage and apoptosis. MC3T3-E1 cells were subjected to EAA starvation, and various assays were conducted to assess cell proliferation, survival, DNA damage, and apoptosis. The antioxidant N-acetylcysteine (NAC) was employed to block ROS formation and mitigate cellular damage. Gene expression and Western blot analyses were performed to elucidate molecular pathways. EAA starvation-induced ROS generation, DNA damage, and apoptosis in MC3T3-E1 cells. NAC administration effectively reduced DNA damage and apoptosis, highlighting the pivotal role of ROS in mediating these cellular responses during EAA deficiency. This study demonstrates that EAA starvation triggers ROS-mediated DNA damage and apoptosis, offering insights into the intricate interplay between nutrient deficiency, oxidative stress, and programmed cell death. NAC emerges as a potential therapeutic intervention to counteract these adverse effects.

A Drosophila model of gestational antimony exposure uncovers growth and developmental disorders caused by disrupting oxidative stress homeostasis.

The toxic heavy metal antimony (Sb) is ubiquitous in our daily lives. Various models have shown that Sb induces neuronal and reproductive toxicity. However, little is known about the developmental toxicity of Sb exposure during gestation and the underlying mechanisms. To study its effects on growth and development, Drosophila stages from eggs to pupae were exposed to different Sb concentrations (0, 0.3, 0.6 and 1.2 mg/mL Sb); RNA sequencing was used to identify the underlying mechanism. The model revealed that prenatal Sb exposure significantly reduced larval body size and weight, the pupation and eclosion rates, and the number of flies at all stages. With 1.2 mg/mL Sb exposure in 3rd instar larvae, 484 genes were upregulated and 694 downregulated compared to controls. Biological analysis showed that the disrupted transcripts were related to the oxidative stress pathway, as verified by reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) and glutathione (GSH) intervention experiments. Sb exposure induced oxidative stress imbalance could be rectified by chelation and antioxidant effects of NAC/GSH. The Drosophila Schneider 2 (S2) model further demonstrated that NAC and GSH greatly ameliorated cell death induced by Sb exposure. In conclusion, gestational Sb exposure disrupted oxidative stress homeostasis, thereby impairing growth and development.

N-Acetylcysteine may exert hepatoprotective effect by regulating Meteorin-Like levels in Adriamycin-induced liver injury.

Adriamycin (ADR) is an important chemotherapeutic drug, but it has serious side effects such as hepatotoxicity. This study aimed to evaluate whether N-acetylcysteine (NAC) has hepatoprotective effects against ADR-induced hepatotoxicity in rats. In addition, it was aimed to determine how Meteorin-Like (MtrnL), which has pleiotropic effects on immunology, inflammation, and metabolism, is affected by ADR and/or NAC applications in liver tissue. 28 rats were randomly assigned to one of four equal groups in the study: control (no treatment), NAC (150 mg/kg/day of NAC intraperitoneally (i.p), ADR (15 mg/kg only on the first day of the experiment), and ADR + NAC (ADR 15 mg/kg on the first day of the experiment + 150 mg/kg/day NAC i.p). After 15 days, liver enzyme levels in serum, oxidant/antioxidant parameters in liver tissue, histopathological changes, caspase 3 (Casp3) and heat shock protein 70 (HSP-70) immunoreactivities, and MtrnL levels were examined. Histopathological changes, liver enzyme levels, as well as HSP-70, and Casp3 immunoreactivities increased due to ADR application. Additionally, MtrnL levels in liver tissue were significantly increased as a result of ADR application. However, it was detected that the NAC application significantly regulated the ADR-induced changes. Furthermore, it was determined that NAC administration regulated the changes in ADR-induced oxidative stress parameters. We propose that NAC may exert a hepatoprotective effect by regulating ADR-induced altered oxidative stress parameters, MtrnL levels, Casp3, and HSP-70 immunoreactivities in the liver.

Reconstructing hepatic metabolic profile and glutathione-mediated metabolic fate of acrylamide.

The toxicity of acrylamide (AA) has continuously attracted wide concerns as its extensive presence from both environmental and dietary sources. However, its hepatic metabolic transformation and metabolic fate still remain unclear. This study aims to unravel the metabolic profile and glutathione (GSH) mediated metabolic fate of AA in liver of rats under the dose-dependent exposure. We found that exposure to AA dose-dependently alters the binding of AA and GSH and the generation of mercapturic acid adducts, while liver as a target tissue bears the metabolic transformation of AA via regulating GSH synthesis and consumption pathways, in which glutamine synthase (GSS), cytochrome P450 2E1 (CYP2E1), and glutathione S-transferase P1 (GSTP1) play a key role. In response to high- and low-dose exposures to AA, there were significant differences in liver of rats, including the changes in GSH and cysteine (CYS) activities and the conversion ratio of AA to glycidamide (GA), and liver can affect the transformation of AA by regulating the GSH-mediated metabolic pathway. Low-dose exposure to AA activates GSH synthesis pathway in liver and upregulates GSS activity and CYS content with no change in γ-glutamyl transpeptidase 1 (GGT1) activity. High-dose exposure to AA activates the detoxification pathway of GSH and increases GSH consumption by upregulating GSTP1 activity. In addition, molecular docking results showed that most of the metabolic molecules transformed by AA and GA other than themselves can closely bind to GSTP1, GSS, GGT1, N-acetyltransferase 8, and dimethyl sulfide dehydrogenase 1. The binding of AA-GSH and GA-GSH to GSTP1 and CYP2E1 enzymes determine the tendentiousness between toxicity and detoxification of AA, which exerts a prospective avenue for targeting protective role of hepatic enzymes against in vivo toxicity of AA.

Structure-guided approach to modify the substrate specificity of the protein human deglycase-1 (hDJ-1).

Glycation is a non-enzymatic reaction wherein sugars or dicarbonyls such as methylglyoxal (MGO) and glyoxal (GO) react with proteins, leading to protein inactivation. The hydrolysing enzyme human deglycase-1 (hDJ-1) is reported to decrease glycative stress by deglycating the modified proteins, specifically at cysteine, lysine, and arginine sites. This specificity of hDJ-1 is thought to be regulated by its active site cysteine residue (Cys106). Structural analysis of hDJ-1 by molecular docking and simulation studies, however, indicates a possible role of glutamate (Glu18) in determining its substrate specificity. To elucidate this, Glu18 present at the catalytic site of hDJ-1 was modified to aspartate (Asp18) by SDM, and the resultant mutant was termed mutant DJ-1 (mDJ-1). Both hDJ-1 and mDJ-1 were heterologously expressed in Escherichia coli BL21 (DE3) strain and purified to homogeneity. The hDJ-1 showed kcat values of 1.45 × 103 s-1, 3.6 × 102 s-1, and 3.1 × 102 s-1, and Km values 0.181 mM, 18.18 mM, and 12.5 mM for N-acetylcysteine (NacCys), N-acetyllysine (NacLys), and N-acetylarginine (NacArg), respectively. The mDJ-1 showed altered kcat values (8 × 102 s-1, 3.8 × 102 s-1, 4.9 × 102 s-1) and Km values of 0.14 mM, 6.25 mM, 5.88 mM for NacCys, NacLys and NacArg, respectively. A single amino acid change (Glu18 to Asp18) improved the substrate specificity of mDJ-1 toward NacLys and NacArg. Understanding hDJ-1's structure and enhanced functionality will facilitate further exploration of its therapeutic potential for the treatment of glycation-induced diabetic complications.

Extraintestinal Manifestations in Induced Colitis: Controversial Effects of N-Acetylcysteine on Colon, Liver, and Kidney.

Ulcerative colitis (UC) is a chronic and recurrent inflammatory bowel disease (IBD) characterized by continuous inflammation in the colonic mucosa. Extraintestinal manifestations (EIM) occur due to the disruption of the intestinal barrier and increased permeability caused by redox imbalance, dysbiosis, and inflammation originating from the intestine and contribute to morbidity and mortality. The aim of this study is to investigate the effects of oral N-acetylcysteine (NAC) on colonic, hepatic, and renal tissues in mice with colitis induced by dextran sulfate sodium (DSS). Male Swiss mice received NAC (150 mg/kg/day) in the drinking water for 30 days before and during (DSS 5% v/v; for 7 days) colitis induction. On the 38th day, colon, liver, and kidney were collected and adequately prepared for the analysis of oxidative stress (superoxide dismutase (SOD), catalase (CAT), glutathione reduced (GSH), glutathione oxidized (GSSG), malondialdehyde (MDA), and hydrogen peroxide (H2O2)) and inflammatory biomarkers (myeloperoxidase (MPO) -, tumor necrosis factor alpha - (TNF-α, and interleukin-10 (IL-10)). In colon, NAC protected the histological architecture. However, NAC did not level up SOD, in contrast, it increased MDA and pro-inflammatory effect (increased of TNF-α and decreased of IL-10). In liver, colitis caused both oxidative (MDA, SOD, and GSH) and inflammatory damage (IL-10). NAC was able only to increase GSH and GSH/GSSG ratio. Kidney was not affected by colitis; however, NAC despite increasing CAT, GSH, and GSH/GSSG ratio promoted lipid peroxidation (increased MDA) and pro-inflammatory action (decreased IL-10). Despite some beneficial antioxidant effects of NAC, the negative outcomes concerning irreversible oxidative and inflammatory damage in the colon, liver, and kidney confirm the nonsafety of the prophylactic use of this antioxidant in models of induced colitis, suggesting that additional studies are needed, and its use in humans not yet recommended for the therapeutic routine of this disease.

N-Acetyl-L-cysteine and aminooxyacetic acid differentially modulate toxicity of the trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine in human placental villous trophoblast BeWo cells.

Trichloroethylene (TCE) is a known human carcinogen with toxicity attributed to its metabolism. S-(1,2-Dichlorovinyl)-L-cysteine (DCVC) is a metabolite of TCE formed downstream in TCE glutathione (GSH) conjugation and is upstream of several toxic metabolites. Despite knowledge that DCVC stimulates reactive oxygen species (ROS) generation and apoptosis in placental cells, the extent to which these outcomes are attributable to DCVC metabolism is unknown. The current study used N-acetyl-L-cysteine (NAC) at 5 mM and aminooxyacetic acid (AOAA) at 1 mM as pharmacological modifiers of DCVC metabolism to investigate DCVC toxicity at concentrations of 5-50 µM in the human placental trophoblast BeWo cell model capable of forskolin-stimulated syncytialization. Exposures of unsyncytialized BeWo cells, BeWo cells undergoing syncytialization, and syncytialized BeWo cells were studied. NAC pre/co-treatment with DCVC either failed to inhibit or exacerbated DCVC-induced H2O2 abundance, PRDX2 mRNA expression, and BCL2 mRNA expression. Although NAC increased mRNA expression of CYP3A4, which would be consistent with increased generation of the toxic metabolite N-acetyl-DCVC sulfoxide (NAcDCVCS), a CYP3A4 inhibitor ketoconazole did not significantly alter BeWo cell responses. Moreover, AOAA failed to inhibit cysteine conjugate ß-lyase (CCBL), which bioactivates DCVC, and did not affect the percentage of nuclei condensed or fragmented, a measure of apoptosis, in all BeWo cell models. However, syncytialized cells had higher CCBL activity compared to unsyncytialized cells, suggesting that the former may be more sensitive to DCVC toxicity. Together, although neither NAC nor AOAA mitigated DCVC toxicity, differences in CCBL activity and potentially CYP3A4 expression dictated the differential toxicity derived from DCVC.